1.Reported by: Marilyn M. Balais Nor-ain L. Barani Madeleine D. Sorino MICROBIAL GROWTH

2. BACTERIAL DIVISION AND GENERATION TIME 3. What is Bacterial Growth? Bacterial Growth - an increase in bacterial numbers - does not refer to an increase in size of the individual cells 4. How to determine Microbial Numbers? directly through counting indirectly through measuring their metabolic activity 5. Bacterial Division Binary Fission - most common method of reproduction, asexual reproduction, splitting of parent cell into two daughter cells Budding - another form of bacterial division, also asexual reproduction, it forms from outgrowths (buds) of mature organisms, it is a form of mitotic cell division, when the bud reaches the size of the parent cell, it separates 6. Binary Fission 7. Filamentous bacteria (some actinomycetes) reproduce by producing chains of conidiospores carried externally at the tips of the filaments. Other filamentous bacteria simply fragment and the fragments initiate the growth of new cells. 8. Generation Time In binary fission, one cells division produces two cells, two cells divisions produces four cells and so on. When the arithmetic number of cells in each generation is expressed as a power of 2 (2x), where x is the exponent that tells the number of doubling (generations) that have occurred. 9. Generation time is the time required for a cell to divide (and its population to double). The generation time among organisms vary according to environmental conditions such as temperature or pH level. Most bacteria have a generation time of 1 3 hours while other species can require up to 24 hours per generation. 10. PHASES OF GROWTH 11. Lag Phase Period of little or no cell division Can last for 1 hour or several days Cells are not dormant Undergoing a period of intense metabolic activity : DNA and enzyme synthesis 12. Log Phase Period of growth also known as logarithmic increase Sometimes called as exponential growth phase Cellular respiration is most active during this period Metabolic activity is active and is most preferable for industrial purposes Sensitive to adverse conditions 13. Stationary Phase Period of equilibrium Metabolic activity of surviving cells slows down which stabilizes the population Cause of discontinuity of exponential growth is not always clear May play a role: exhaustion of nutrients, accumulation of waste products and harmful changes in pH Chemostat continuous culture used in industrial fermentation 14. Death Phase Also known as Logarithmic Decline Phase Continues until a small fraction of the population is diminished Some population dies out completely Others retain surviving cells indefinitely while others only retain for a few days 15. Direct Measurement of Microbial Growth 16. Plate Counts Measures the number of viable cells It takes about 24 hours or more for visible colonies to form Reported as colony-forming units (CFU) Only a limited number of colonies must be developed in the plate because when too many colonies are present, some cells are overcrowded and do not develop. The original inoculum is diluted several times in a process called serial dilution to ensure that colony counts will be within 25 250 colonies. 17. Serial Dilutions Example: A milk sample has 10,000 bacteria per milliliter. If 1 ml of this sample were plated out, there would theoretically be 10,000 colonies formed in the Petri plate of the medium. Obviously, this would not produce a countable plate. If 1 ml of this sample were transferred to a tube containing 9 ml of sterile water, each milliliter of fluid in this tube would now contain 1000 bacteria. If 1 ml of this sample were inoculated into a Petri plate, there would still be too many potential colonies to count on a plate. Therefore, another serial dilution could be made. 18. One milliliter containing 1000 bacteria would be transferred to a second tube of 9 ml of water. Each milliliter of this tube would now contain only 100 bacteria, and if 1 ml of the contents of this tube were plated out, potentially 100 colonies would be formed an easily countable number. 19. Plate counts and serial dilutions 20. Pour Plates and Spread Plates A plate count is done by either the pour plate method or the spread plate method 21. Methods of preparing plates for plate counts 22. Pour Plates: Disadvantages Some relatively heat-sensitive microorganisms may be damaged by the melted agar and will then be unable to form colonies When certain differential media are used, the distinctive appearance of the colony on the surface is essential for diagnostic purposes. Colonies that form beneath the surface of a pour plate are not satisfactory for such tests. To avoid these problems, the spread plate method is used instead 23. Filtration Used when the quantity of bacteria is very small 24. PHYSICAL REQUIREMENTS 25. Temperature Psychrophiles - cold-loving microbes about -10C to 20C optimum growth 15C not grow in 25C Mesophiles - moderate-temperature- loving microbes about 10C to 50C optimum growth 25C to 40C Thermophiles - heat-loving microbes about 40C to 70C optimum growth 50C to 60C 26. Psychrotrophs - microorganisms responsible for spoilage of refrigerated food about 0C to 30C Hyperthermophiles/Extreme Thermophiles about 65C to 110C optimum growth 80C *usually has 30C between maximum and minimum growth 27. pH Acidity or alkalinity of a solution Most bacteria grow best at pH 6.5-7.5 Very few bacteria grow below pH 4 Acidophiles - chemoautotrophic bacteria that are remarkably tolerant of acidity 28. Osmotic Pressure High osmotic pressures have the effect of removing necessary water from a cell Plasmolysis - shrinkage of cells plasma membrane caused by osmotic loss of water 29. Plasmolysis 30. Extreme Halophiles - adapted well to high salt concentrations Obligate Halophiles - require high salt concentrations for growth Facultative Halophiles - do not require high salt concentrations but are able to grow at salt concentrations up to 2%, some can tolerate at 15% salt 31. CHEMICAL REQUIREMENTS 32. Carbon Structural backbone of living matter, it is needed for all organic compounds to make up a living cell Chemoheterotrophs get most of their carbon from the source of their energy---organic materials such as proteins, carbohydrates and lipids Chemoautotrophs and photoautotrophs derive their carbon from carbon dioxide 33. Nitrogen, Sulfur and Phosphorus For synthesis of cellular material Nitrogen and sulfur is needed for protein synthesis Nitrogen and phosphorus is needed for syntheses of DNA, RNA and ATP Nitrogen- 14% dry weight of a bacterial cell Sulfur and phosphorus- 4% 34. Trace Elements Microbes require very small amounts of other mineral elements, such as Fe, Cu, Mo, Zn Essential for certain functions of certain enzymes Assumed to be naturally present in tap water and other components of media 35. Oxygen Obligate Aerobes - only aerobic growth, oxygen required, growth occurs with high concentration of oxygen Facultative Aerobes - both aerobic and anaerobic growth, greater growth in presence of water, growth is best in presence of water but still grows without presence of oxygen 36. Obligate Anaerobe - only anaerobic growth, growth ceases in presence of oxygen, growth occurs only when there is no oxygen Aerotolerate Anaerobe - only anaerobic growth, but continues in presence of oxygen, oxygen has no effect Microaerophiles - only aerobic growth, oxygen required in low concentration, growth occurs only where a low concentration of oxygen has diffused into medium 37. Organic Growth Factors Essential organic compounds an organism is unable to synthesize, they must be directly obtained from the environment Some bacteria lack the enzymes needed for synthesis for certain vitamins, so they must obtain them directly Examples: amino acids, purines, pyrimidines 38. CULTURE MEDIA 39. Culture Media (an overview) Culture Medium a nutrient material prepared for the growth of microorganisms in a laboratory Inoculum when microbes are introduced into a culture medium to initiate growth Sterile initially containing no living organisms Agar a complex polysaccharide derived from a marine alga which has long been used as a thickener in foods such as jellies and ice cream 40. Cont Slants what test tubes are called when agar is allowed to solidify with the tube held at an angle so that a large surface area for growth is available Deep what test tubes are called when the agar solidifies vertically within the tube Petri (or culture) plates what Petri dishes are called when filled 41. Chemically Defined Media One which the exact chemical composition is known Used for laboratory experimental work or autotrophic bacteria 42. Complex Media Made up of nutrients from extracts of yeasts, meat, plants or digest of protein Examples are nutrient broth and nutrient agar 43. Anaerobic Growth Media and Methods Must use reducing media that contain chemicals like sodium thioglycolate that combine with oxygen to deplete it Labs may have special incubators for anaerobes or capnophiles (microbes that grow better with increased carbon dioxide) 44. A jar for cultivating anaerobic bacteria on Petri plates 45. Special Culture Techniques Carbon Dioxide Incubators Candle Jars Small plastic bags with self-contained chemical gas generators 46. An anaerobic chamber 47. Candle Jar 48. Selective Media Used to suppress the growth of unwanted bacteria and encourage the growth of desired microbes Example: Sabourads dextrose agar which has a pH of 5.6 is used to isolate fungi because of pH. bismuth sulfite agar isolates the typhoid bacterium 49. Sabourads Dextrose Agar 50. Differential Media Provides nutrients and environmental conditions that favor the growth of a particular microbe. To increase the number of a microbes to prevent missing a microbe that may be in small numbers. Often used on soil or fecal samples. Example: soil sample looking for bacteria that grow on phenol. 51. Blood agar, a differential medium containing red blood cells. 52. Bacterial colonies on several differential media 53. Differential Media 54. Selective and Differential Media Sometimes there are media that are both selective and differential. Examples: MacConkey agar Mannitol salt agar 55. Mannitol Salt agar inoculated with Staphylococcus aureus, Staphylococcus epidermidis and Micrococcus. S.aureus is able to ferment mannitol, creating acidic byproducts which turn the pH indicator in the agar yellow. S.epidermidis is unable to ferment mannitol and so creates alkaline byproducts, turning the pH indicator pink. Micrococcus cannot grow on this medium and so produces no reaction. 56. THANK YOU FOR LISTENING!!!